The goal of this project is the development of assays and devices that will enable rapid nucleic acid-based diagnosis of human pathogens in a simple, inexpensive, and user-friendly format. Such a system will allow health care professionals in a point-of-care setting to identify and treat infectious diseases in a timely manner, thereby improving patient outcomes and preventing further spread. The project focuses on Herpes Simplex Virus type 1 and 2 (HSV-1 and HSV-2), which can lead to life-threatening infections in newborns and immuno- compromised individuals. A need exists for rapid HSV diagnosis in STD clinics and in maternity wards. The project involves a multidisciplinary research team with expertise in bioassay development, engineering and clinical pathology. This team will develop a system which shall be capable of type-common or type-specific HSV detection from swab samples of herpetic lesions (reported to contain on average 8*104 virus particles per mL lysis buffer), with no interference from background human DNA or from other bacterial or viral pathogens associated with orogenital lesions. The system shall employ a disposable and self-contained sample-in answer-out cartridge format that mates to a low cost handheld electronics module, to provide ease-of-use and to avoid cross-contamination. The total assay time including sample preparation and detection shall be less than 30 minutes. The project builds on previous work combining novel isothermal DNA amplification reactions with colorimetric detection of oligonucleotides through DNA-functionalized gold nanospheres, and on the development of microfluidic systems for biothreat detection. The first aim of this project is to further develop and systematically optimize single- and multiplex assays for rapid, sensitive, and robust detection of HSV from DNA isolates of clinical samples, involving isothermal amplification followed by visual read-out. The second aim is to establish and optimize simple but efficient sample preparation protocols suitable for coupling to the isothermal assays developed under aim 1, to facilitate robust HSV detection from swab samples of herpetic lesions. The third aim focuses on developing device prototypes to execute the sample preparation and isothermal amplification methods developed under aims 1 and 2. This project presents a unique opportunity to bring together multidisciplinary expertise to develop a next generation of assays and analyzers for nucleic acid testing. The envisioned system will enable rapid pathogen detection in point of care settings with the same robustness and sensitivity as current (mostly PCR-based) methods, while reducing the cost by 1-2 orders of magnitude compared to current benchtop systems for nucleic acid testing at the point of care. In the long term, the assays and devices developed herein can be applied to other clinical pathogens, including RNA viruses such as HIV and influenza, to biothreat detection, and to resource-limited settings.